Enzymatic Cleavage of 3\u2019-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis

Shiuan-Woei Linwu,Hung-Wen Chi,Hsiang-Ming Wang,Yu-Ping Tsao,Ya-Chen Chen,Chung-Fan Chiou,Cheng-Yao Chen,Yu-Hsuan Tu,Wei-Hsin Chang,Johnsee Lee,Ting-Yueh Tsai

doi:10.1038/s41598-020-64541-z

Abstract

The reversible dye-terminator (RDT)-based DNA sequencing-by-synthesis (SBS) chemistry has driven the advancement of the next-generation sequencing technologies for the past two decades. The RDT-based SBS chemistry relies on the DNA polymerase reaction to incorporate the RDT nucleotide (NT) for extracting DNA sequence information. The main drawback of this chemistry is the “DNA scar” issue since the removal of dye molecule from the RDT-NT after each sequencing reaction cycle leaves an extra chemical residue in the newly synthesized DNA. To circumvent this problem, we designed a novel class of reversible (2-aminoethoxy)-3-propionyl (Aep)-dNTPs by esterifying the 3’-hydroxyl group (3’-OH) of deoxyribonucleoside triphosphate (dNTP) and examined the NT-incorporation activities by A-family DNA polymerases. Using the large fragment of both Bacillus stearothermophilus (BF) and E. coli DNA polymerase I (KF) as model enzymes, we further showed that both proteins efficiently and faithfully incorporated the 3’-Aep-dNMP. Additionally, we analyzed the post-incorporation product of N + 1 primer and confirmed that the 3’-protecting group of 3’-Aep-dNMP was converted back to a normal 3’-OH after it was incorporated into the growing DNA chain by BF. By applying all four 3’-Aep-dNTPs and BF for an in vitro DNA synthesis reaction, we demonstrated that the enzyme-mediated deprotection of inserted 3’-Aep-dNMP permits a long, continuous, and scar-free DNA synthesis.

Highlights

The next-generation sequencing (NGS) technologies have revolutionized modern biomedical research
To elucidate the intrinsic features of A-family DNA polymerases (AF-DNAPs) and deduce how AF-DNAPs could readily accept and incorporate the 3’-esterified nucleotide (NT), we first built the computational model of BF ternary complex with a primer-template DNA and a 3’-esterified nucleotide based on the existing ternary structure of BF (PDB file:1LV5)[31] (Fig. 2C)
By applying all four 3’-Aep-deoxyribonucleoside triphosphate (dNTP) for an in vitro DNA synthesis reaction, we demonstrated that the A-family DNA polymerases can efficiently insert 3’-Aep-dNMPs and permit a long, continuous, and scar-free DNA synthesis

Summary

Introduction

The next-generation sequencing (NGS) technologies have revolutionized modern biomedical research. Except for the pyrosequencing[11,12] and semiconductor-based proton-sequencing techniques[13], in which both directly use the normal nucleoside triphosphate (dNTP) for DNA sequencing reactions, the nucleotide (NT) substrates adopted by the mainstream SBS-driven NGS platforms, including Illumina, Qiagen, and Pacific Biosciences (PacBio), are extensively modified In both Illumina’s and Qiagen’s sequencing chemistry, the purine (A or G) or pyrimidine (C or T) base of nucleotide (NT) is individually attached with a distinct spectrum of fluorescent dye for signal detection. The cycle of NT addition by DNA Pol can resume and the growing DNA chain can further be extended In this reversible SBS reaction, the removal of fluorescent dye from the NT after each reaction cycle leaves an extra, chemical linker moiety, which covalently connects the dye molecule to the base, on the normal purine or pyrimidine NTs in the newly synthesized DNA. Using the large fragment of Bacillus stearothermophilus DNA polymerase I (BF) as a model enzyme, we tested the feasibility of applying these 3’-esterified dNTPs for an uninterrupted and scar-free DNA synthesis

Methods

Results

Conclusion